1. Field of the Invention
The present invention relates to high throughput assay systems and methods for identifying agents that alter the level of surface expression of integral membrane proteins, such as cardiac ion channels, in mammalian cells. The present invention also relates to therapeutic methods of using agents identified using such methods.
2. Background of the Related Art
A. Assays
Human Ether-a-go-go Related Gene (HERG) is the pore-forming potassium channel subunit that underlies the cardiac repolarizing current IKr and consists of six transmembrane segments (S1–S6) and cytoplasmic amino- and carboxyl-termini. HERG has been linked to both congenital and drug-induced long QT syndrome, a serious and potential fatal heart condition.
Mutations in hERG produce functionally impaired channels and/or trafficking defective channels, both of which reduce IKr currents. Mutations spanning most of the molecule have been identified in different long QT families. This suggests that hERG plays a critical role in cardiac physiology.
Most of the drugs associated with long QT syndrome (drug-induced) are hERG blockers. See, e.g., Vandenberg et al, Trends Pharmacol Sci. 22:240–246 (2001). Since the cardiotoxicity of the non-sedating antihistamine terfenadine (Seldane) was linked to hERG block in 1996 (see Roy et al, Circulation 94:817–823 (1996)), a wide variety of drugs having diverse structures, including antiarrhythmics, antibiotics, antipsychotics as well as antihistamines, have been shown to be potent hERG blockers.
Accordingly, hERG has become an important target for cardiac safety testing of new therapeutic agents. The US Food & Drug Administration currently recommends that pharmaceutical companies seeking approval for novel therapeutic compounds have them screened for potential hERG blocking.
Presently, hERG cardiac safety testing involves eletrophysiology and consits of patch clamp recording of hERG currents in HEK 293 cells which stably overexpress hERG. This assay, however, is expensive, time-consuming and requires considerable expertise. Consequently, it is usually done relatively late in the drug development process. Unfortunately, at that time, discovery that a novel therapeutic compound is a potent hERG blocker would be potentially devastating to the prospects of that compound being approved and used therapeutically. As a result, there is considerable interest in the pharmaceutical industry for assays for hERG blockers that are both less expensive and faster, and that can be employed much earlier in the drug development process.
The limitations of the patch clamp assay has led to alternative methods for preclinical screening of drugs for potential hERG interactions. Several methods have been described, but are limited, for example, in sensitivity, throughput capacity and/or false-postive rates (see Xu et al., Drug Discover Today 6:1278–1287 (2001)).
For example, one type of assay uses membrane potential sensitive fluorescent dyes, such as DiBAC4 or FMP. Since these assays measure changes in membrane potential and not hERG activity, the risk of false positives (i.e. drugs which change membrane potential but do not block hERG) is great. A recent evaluation of such assays (fang et al., J. Biomol. Screen. 6:325–331 (2001)) indicates a signficant problem with false positives and, to a lesser extent, false negatives. In addition, sensitivity is reduced about 100-fold. Moreover, the rank order of hERG blocker potency differs with membane potential assays relative to patch clamp measurements, limiting the use of such fluorescent assays to identifying potential hERG channel blockers without providing useful information as to their potency. Finally, dye/drug interactions have also caused problems in this assay.
A second assay suggested as a potential high throughput preclinical screen for hERG interactions is [3H]-dofelitide binding to membranes for hERG transfected cells. (See Finlayson etaz., Eur. J. Pharm 430:147–148 (2001)). This binding assay is nonfunctional and relies on the ability of drugs to compete with [3H]-dofelitide for binding to hERG channels. In preliminary experiments, however, the rank order of hERG blockers identified by patch clamp methods was not replicated in the [3H]-dofelitide binding assay. Also, the requirements for purified cell membranes as binding substrate and radio-labelled dofelitide limit the usefulness of this assay.
A third assay that has been suggested involves the measurement of rubidium (Rb) flux through cells expressing hERG (see Terstappen, Anal Biochem. 272:149–155 (1999)). These cells ate loaded with Rb channels and activated with high potassium levels, and Rb released into the medium is measured. The rank order of potency obtained by this method, however, does not correlate with patch clamp data (see Tang et al, J. Biomol. Screen., 6:325–331 (2001)). In addition, throughput is limited and sensitivity reduced 10-fold.
Accordingly, there remains a need for assay systems for identifying blockers of integral membrane proteins, including cardiac ion channels such as hERG.
B. Cardiac Arrhythmias
Atrial flutter and/or atrial fibrillation (AF) are the most commonly sustained cardiac arrhythrnlas in clinical practice, and are likely to increase in prevalence with the aging of the population. Currently, AF affects more than 1 million Americans annually, represents over 5% of all admissions for cardiovascular diseases and causes more than 80,000 strokes each year in the United States. While AF is rarely a lethal arrhythmia, it is responsible for substantial morbidity and can lead to complications such as the development of congestive heart failure or thromboembolism. Currently available Class I and Class III anti-arrhythmic drugs reduce the rate of recurrence of AF, but are of limited use because of a variety of potentially adverse effects, including ventricular proarrhythmia. Because current therapy is inadequate and fraught with side effects, there is a clear need to develop new therapeutic approaches.
Ventricular fibrillation (VF) is the most common cause associated with acute myocardial infarction, ischemic coronary artery disease and congestive heart failure. As with AF, current therapy is inadequate and there is a need to develop new therapeutic approaches.
Although various anti-arrhythmic agents are now available on the market, those having both satisfactory efficacy and a high margin of safety have not been obtained. For example, anti-arrhythmic agents of Class I, according to the classification scheme of Vaughan-Williams (“Classification of antiarrhythmic drugs”, Cardiac Arrhythmias, edited by: E. Sandoe, E. Flensted-Jensen, K. Olesen; Sweden, Astra, Sodertalje, pp 449–472 (1981)), which cause a selective inhibition of the maximum velocity of the upstroke of the action potential (Vmax) are inadequate for preventing ventricular fibrillation because they shorten the wave length of the cardiac action potential, thereby favoring re-entry. In addition, they have problems regarding safety, i.e. they cause a depression of myocardial contractility and have a tendency to induce arrhythmias due to an inhibition of impulse conduction. The CAST (coronary artery suppression trial) study was terminated while in progress because the Class I antagonists had a higher mortality than placebo controls. β-adrenergenic receptor blockers and calcium channel (ICa) antagonists, which belong to Class II and Class IV, respectively, have a defect in that their effects are either limited to a certain type of arrhythmia or are contraindicated because of their cardiac depressant properties in certain patients with cardiovascular disease. Their safety, however, is higher than that of the anti-arrhythmic agents of Class I.
Anti-arrhythric agents of Class III are drugs that cause a selective prolongation of the action potential duration (APD) without a significant depression of the maximum upstroke velocity (Vmax). They therefore lengthen the save length of the cardiac action potential increasing refractories, thereby antagonizing re-entry. Available drugs in this class are limited in number. Examples such as sotalol and ariodarone have been shown to possess interesting Class III properties (Singh B. N., Vaughan Williams E. M., “A third class of anti-arrhythmic action: effects on atrial and ventricular intracellular potentials and other pharmacological actions on cardiac muscle of MJ 1999 and AH 3747”, Br. J. Pharmacol 39:675–689 (1970), and Singh B. N., Vaughan Williams E. M., “The effect of amiodarone, a new anti-anginal drug, on cardiac muscle”, Br. J. Pharinacol 39:657–667 (1970)), but these are not selective Class III agents.
Sotalol also possesses Class II (β-adrenergic blocking) effects which may cause cardiac depression and is contraindicated in certain susceptible patients.
Amiodarone also is not a selective Class III antiarrhythmic agent because it possesses multiple electrophysiological actions and is severely limited by side effects. (Nademanee, K., “The Amiodarone Odyssey”, J. Am. Coll. Cardiol. 20:1063–1065 (1992)) Drugs of this class are expected to be effective in preventing ventricular fibrillation. Selective Class III agents, by definition, are not considered to cause myocardial depression or an induction of arrhythmias due to inhibition of conduction of the action potential as seen with Class I antiarrhythmic agents.
Class III agents increase myocardial refractoriness via a prolongation of cardiac action potential duration (APD). Theoretically, prolongation of the cardiac action potential can be achieved by enhancing inward currents (i.e. Na+ or Ca2+ currents; hereinafter INa and ICa, respectively) or by reducing outward repolarizing potassium K+ currents. The delayed rectifier (IK)K+ current is the main outward current involved in the overall repolarization process during the action potential plateau, whereas the transient outward (Ito) and inward rectifier (IKI)K+ currents are responsible for the rapid initial and terminal phases of repolarization, respectively.
Cellular electrophysiologic studies have demonstrated that IK consists of two pharmacologically and kinetically distinct K+ current subtypes, IKr (rapidly activating and deactivating) and IKs (slowly activating and deactivating). (Sanguinetti and Jurkiewicz, “Two components of cardiac delayed rectifier K+ current. Differential sensitivity to block by Class III anti-arrhythmic agents”, J Gen Physiol 96:195–215 (1990)). IKr is also the product of the human ether-a-go-go gene (hERG). Expression of hERG cDNA in cell lines leads to production of the hERG current which is almost identical to IKr (Curran et al., “A molecular basis for cardiac arrhythmia: hERG mutations cause long QT syndrome,” Cell 80(5):795–803 (1995)).
Class III anti-arrhythmic agents currently in development, including d-sotalol, dofetilide (UK-68,798), almokalant (H234/09), E-4031 and methanesulfonamide-N-[1′-6-cyano-1,2,3,4-tetrahydro-2-naphthalenyl)-3,4-dihydro-4-hydroxyspiro[2H-1-benzopyran-2,4′-piperidin]-6yl], (+)-, monochloride (MK-499) predominantly, if not exclusively, block IKr. Although, amiodarone is a blocker of IKs (Balser J. R. Bennett, P. B., Hondeghem, L. M. and Roden, D. M. “Suppression of time-dependent outward current in guinea pig ventricular myocytes: Actions of quinidine and amiodarone”, Circ. Res. 69:519–529 (1991)), it also blocks INa and ICa, effects thyroid function, is as a nonspecific adrenergic blocker, acts as an inhibitor of the enzyme phospholipase, and causes pulmonary fibrosis (Nademanee, K. “The Amiodarone Odessey”. J. Am. Coll. Cardiol. 20:1063–1065 (1992)).
Reentrant excitation (reentry) has been shown to be a prominent mechanism underlying supraventricular arrhythmias in man. Reentrant excitation requires a critical balance between slow conduction velocity and sufficiently brief refractory periods to allow for the initiation and maintenance of multiple reentry circuits to coexist simultaneously and sustain AF. Increasing myocardial refractoriness by prolonging APD, prevents and/or terminates reentrant arrhythmias. Most selective, Class III antiarthythmic agents currently in development, such as d-sotalol and dofetilide predominantly, if not exclusively, block IKr, the rapidly activating component of IK found both in atrium and ventricle in man.
Since these IKr blockers increase APD and refractoriness both in atria and ventricle without affecting conduction per se, theoretically they represent potential useful agents for the treatment of arrhythmias like AF and VF. These agents have a liability in that they have an enhanced risk of proarrhythmia at slow heart rates. For example, torsade de pointes, a specific type of polymorphic ventricular tachycardia which is commonly associated with excessive prolongation of the electrocardigraphic QT interval, hence termed “acquired long QT syndrome”, has been observed when these compounds are utilized (Roden, D. M. “Current Status of Class III Antiarrhythrnic Drug Therapy”, Am J. Cardiol, 72:44B–49B (1993)). The exaggerated effect at slow heart rates has been termed “reverse frequency-dependence” and is in contrast to frequency-independent or frequency-dependent actions. (Hondeghem, L. M., “Development of Class III Antiarrhythmic Agents”, J. Cardiovasc. Cardiol. 20 (Suppl. 2):S17–S22). The pro-arrhythmic tendency led to suspension of the SWORD trial when d-sotalol had a higher mortality than placebo controls.
The slowly activating component of the delayed rectifier (IKs) potentially overcomes some of the limitations of IKr blockers associated with ventricular arrhythmias. Because of its slow activation kinetics, however, the role of IKs in atrial repolarization may be limited due to the relatively short APD of the atrium. Consequently, although IKs blockers may provide distinct advantage in the case of ventricular arrhythmias, their ability to affect supra-ventricular tachyarrhythmias (SVT) is considered to be minimal.
Another major defect or limitation of most currently available Class III anti-arrythmic agents is that their effect increases or becomes more manifest at or during bradycardia or slow heart rates, and this contributes to their potential for proarrhythmia. On the other hand, during tachycardia or the conditions for which these agents or drugs are intended and most needed, they lose most of their effect. This loss or diminishment of effect at fast heart rates has been termed “reverse use-dependence” (Hondeghem and Snyders, “Class III antiarrhythmic agents have a lot of potential but a long way to go: Reduced effectiveness and dangers of reverse use dependence”, Circulation, 81:686–690 (1990); Sadanaga et al., “Clinical evaluation of the use-dependent QRS prolongation and the reverse use-dependent QT prolongation of class III anti-arrhythmic agents and their value in predicting efficacy” Amer. Heart Journal 126:114–121 (1993)), or “reverse rate-dependence” (Bretano, “Rate dependence of class III actions in the heart”, Fundam. Clin. Pharmacol. 7:51–59 (1993); Jurkiewicz and Sanguinetti, “Rate-dependent prolongation of cardiac action potentials by a methanesulfonanilide class III anti-arrhythmic agent: Specific block of rapidly activating delayed rectifier K+ current by dofetilide”, Circ. Res. 72:75–83 (1993)). Thus, an agent that has a use-dependent or rate-dependent profile, opposite that possessed by most current class III anti-arrhythmic agents, should provide not only improved safety but also enhanced efficacy.
In view of the problems associated with current class III anti-arrhythmic agents, there remains a need for an effective treatment of cardiac arrhythrmias in mammals.